Diabetes typing marker based on D2D3-TROY signal axis and application

Abstract

The invention discloses a diabetes typing marker based on a D2D3-TROY signal axis, a new medical application of valprophylline and a kit for diagnosing a specific diabetes subtype.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to novel molecular subtyping biomarkers for diabetes, and new uses of the small molecule compound propentofylline (PPF) in the prevention and / or treatment of specific diabetes subtypes. Background Technology

[0002] Diabetes mellitus is a prevalent chronic metabolic disease worldwide, characterized by hyperglycemia resulting from insufficient insulin secretion and / or impaired insulin action. China has approximately 141 million people aged 20-79 with the disease, ranking first globally. Current clinical classifications (type 1, type 2, gestational diabetes, etc.) fail to fully reflect individual differences among patients, leading to significant variations in treatment response, complication risk, and prognosis within the same classification, thus failing to meet the needs of precision medicine.

[0003] Pancreatic β-cell dysfunction (leading to insufficient insulin secretion) and reduced β-cell numbers (due to decreased proliferative capacity) are core pathological links in the development and progression of diabetes, but their precise molecular regulatory mechanisms are not yet fully elucidated. Transcription factors such as MafA, PDX-1, and Nkx6.1 are known to be crucial for maintaining β-cell function and proliferation. Studies have shown that specific knockout of Kindlin-2 in β-cells leads to decreased MafA protein levels, inhibiting normal insulin secretion and resulting in diabetes. Furthermore, the Wnt / β-Catenin and PDGFRα-Ezh2 signaling pathways have been confirmed to play important roles in regulating β-cell proliferation.

[0004] D2D3 is a circulating factor produced by the cleavage of uPAR protein. It has been identified as a pathogenic factor for a specific subtype of insulin-dependent diabetes mellitus. It can significantly impair the insulin secretion function of β cells and inhibit their proliferative capacity.

[0005] Current conventional treatments for diabetes have significant limitations: long-term exogenous insulin therapy may cause a series of adverse reactions, such as weight gain, risk of hypoglycemia, worsening insulin resistance, and potential cardiovascular side effects; while islet transplantation helps patients break free from long-term dependence on exogenous insulin, it faces the constraint of a severe shortage of donors, and the inherent proliferative capacity of mature β cells is extremely low (only about 0.2% have the potential to divide); some small molecule compounds designed to promote β cell proliferation (such as Harmine, LY364947, etc.) have the risk of "off-target effects" and systemic adverse reactions due to their relatively broad target range; although D2D3 neutralizing antibodies have shown potential in restoring β cell function, their high production cost, complex in vivo pharmacokinetic behavior, and limited tissue penetration limit their clinical application.

[0006] The small molecule compound propargyl theophylline (PPF) is an atypical synthetic methylxanthine derivative with multi-target action characteristics, and has attracted attention, especially in the treatment of central nervous system diseases and tumors. (1) In the field of central nervous system, propargyl theophylline has completed phase II and phase III clinical trials for Alzheimer's disease and vascular dementia. Its neuroprotective mechanism mainly includes: ① by specifically reducing the expression of TROY protein in microglia, inhibiting the activation of downstream pro-inflammatory signaling pathways, thereby reducing neuroinflammatory response and neuronal damage; ② exerting a mild cerebral vasodilatory effect, improving cerebral blood flow perfusion (especially for ischemic areas), while improving the efficiency of brain tissue in utilizing glucose and oxygen, optimizing energy metabolism, and alleviating ischemia and hypoxia-related cognitive dysfunction; ③ propargyl theophylline has natural blood-brain barrier penetration ability and can achieve effective concentrations in the central nervous system without the need for a complex drug delivery system. (2) In the field of tumor research, especially in glioblastoma models, theophylline can inhibit the invasiveness and drug resistance of tumor cells by intervening in the TROY-related signaling pathway. In addition, theophylline can also significantly enhance the sensitivity of tumor cells to temozolomide (TMZ, the standard chemotherapy drug for glioma) and radiotherapy.

[0007] The above-mentioned existing technologies demonstrate that theophylline has a regulatory effect on the TROY signaling pathway, providing important evidence for its application in drug repurposing research.

[0008] TROY (also known as TNFRSF19) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). The applicant's preliminary exploratory studies found that propargyl theophylline can significantly inhibit D2D3-induced upregulation of TROY protein expression, thereby effectively reversing the inhibitory effect of D2D3 on insulin secretion. However, whether a direct ligand-receptor relationship exists between D2D3 and TROY, the specific molecular mechanism by which the D2D3-TROY signaling axis regulates β-cell function, and whether PPF can serve as a diabetes treatment targeting this signaling axis are all unresolved and urgently need to be addressed in this field.

[0009] Therefore, it is urgent to systematically elucidate the specific molecular mechanisms by which the D2D3-TROY potential signaling axis regulates insulin secretion and β-cell proliferation, and on this basis, to provide a therapeutic strategy that targets this signaling axis. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art, to reveal for the first time the core role of D2D3-TROY as a new signaling axis in the occurrence and development of diabetes, especially certain subtypes of diabetes, and to develop new biomarkers, kits and new pharmaceutical uses of valproic acid based on this.

[0011] A first aspect of the invention provides the use of the TROY protein as a biomarker in the preparation of tools for diagnosing D2D3-related diabetes subtypes.

[0012] Furthermore, in the first aspect, the diagnosis includes: comparing the expression level or activity level of the TROY protein in a subject's sample with a reference value, wherein an elevated level indicates that the subject has the diabetes.

[0013] Furthermore, in the first aspect, the D2D3-related diabetes subtype is characterized by: the presence of specific binding of D2D3 to TROY in the patient's body, and / or increased TROY expression or activity mediated by D2D3.

[0014] A second aspect of the invention provides the use of pentyl theophylline or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof in the preparation of a medicament for the prevention and / or treatment of diabetes.

[0015] Furthermore, in the second aspect, the diabetes is a D2D3-related diabetes subtype.

[0016] Furthermore, in the second aspect, the D2D3-related diabetes subtype is characterized by: the presence of specific binding of D2D3 to TROY in the patient's body, and / or increased TROY expression or activity mediated by D2D3.

[0017] Preferably, the theophylline exerts its therapeutic effect by downregulating TROY protein levels.

[0018] Preferably, the theophylline is used to restore the insulin secretion function and / or proliferative capacity of pancreatic β cells.

[0019] Furthermore, the restoration of insulin secretion function is related to the upregulation of MafA transcription factor expression or activity, and the restoration of proliferation capacity is related to the activation of the PDGFRα-Ezh2 signaling pathway.

[0020] A third aspect of the present invention provides a kit for diagnosing D2D3-related diabetes mellitus subtypes, the kit comprising reagents for detecting the expression and / or activity levels of TROY protein in a sample.

[0021] Furthermore, in the third aspect, the reagent comprises an antibody and / or its antigen-binding fragment capable of specifically binding to the TROY protein.

[0022] A fourth aspect of the present invention provides a kit for precision medicine of diabetes, comprising: (a) a reagent for detecting the expression level of TROY protein in a sample; and (b) pentylene or a pharmaceutically acceptable salt thereof.

[0023] A fifth aspect of the invention provides a pharmaceutical composition for the prevention and / or treatment of diabetes, comprising: a therapeutically effective amount of theophylline or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof; and a pharmaceutically acceptable carrier.

[0024] Furthermore, in the fifth aspect, the diabetes is a D2D3-related diabetes subtype.

[0025] like Figure 1 As shown, the overall mechanism by which the D2D3-TROY signaling axis regulates β-cell function and proliferation in this invention is as follows: the blood-circulating factor D2D3 specifically binds to the β-cell surface receptor TROY, triggering: 1) the intracellular domain of TROY is cleaved and enters the cell nucleus, interacting with MafA and affecting MafA stability; or 2) the intracellular domain of TROY interacts with Kindlin-2, reducing the protein level and nuclear translocation level of Kindlin-2, thereby affecting MafA stability through Kindlin-2; 3) TROY inhibits the activation of the PDGFRa-Ezh2 signaling pathway by PDGF-AA.

[0026] Beneficial effects

[0027] 1. Pioneering discovery: For the first time in the field of diabetes, it has been confirmed that TROY is a functional receptor for D2D3, and that the D2D3-TROY signaling axis is the core mechanism leading to a specific subtype of diabetes, filling a knowledge gap in this field.

[0028] 2. Precision Diagnostic Value: Based on the above findings, TROY has been established as a specific molecular marker for this type of diabetes subtype (i.e., D2D3-related diabetes subtype), providing a new tool and basis for the precise classification and diagnosis of diabetes.

[0029] 3. Repurposing an Existing Drug with Promising Translational Prospects: This invention creatively proposes a novel use for theophylline, a drug with a proven track record of safe human use, in the treatment of D2D3-related diabetes subtypes. This invention demonstrates that PPF exhibits high specificity in regulating the TROY protein, avoiding the unintended side effects caused by the "off-target effects" of traditional small molecule compounds. PPF can specifically downregulate the D2D3-TROY signaling axis, restoring β-cell function, and its effectiveness has been verified at the cellular level. This approach avoids the lengthy and high-risk nature of new drug development and possesses enormous potential for rapid clinical translation.

[0030] 4. Complete Mechanism Elucidation: The system elucidates the complete molecular mechanism by which the D2D3-TROY signaling axis impairs β-cell function and proliferation through a dual pathway of "inhibiting the Kindlin-2-MafA pathway" and "blocking the PDGFRα-Ezh2 pathway," providing a theoretical basis for subsequent drug development. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the overall mechanism by which the D2D3-TROY signaling axis regulates β-cell function and proliferation according to the present invention.

[0032] Figure 2 Figure showing the construction of the D2D3-Tg mouse model and the effect of D2D3 on increasing the level of TROY protein in β cells.

[0033] Figure 3 This is a verification diagram showing the direct interaction between D2D3 and TROY.

[0034] Figure 4 A graph showing the binding kinetics of D2D3 and TROY determined using SPR technology.

[0035] Figure 5 This figure shows the specific expression of TROY in β cells and its functional relationship with D2D3.

[0036] Figure 6 This diagram validates the mechanism by which the D2D3-TROY signaling axis inhibits β-cell proliferation.

[0037] Figure 7 This is a verification diagram showing that propargyl theophylline (PPF) inhibits D2D3-mediated TROY expression and restores insulin secretion. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0039] The materials used in the following embodiments are from the following sources:

[0040] D2D3 protein: obtained by plasmid transfection and purification in HEK293T cells;

[0041] TROY protein: obtained by plasmid transfection and purification in HEK293T cells;

[0042] HEK293T cells: This cell line was obtained commercially.

[0043] Example 1: Verification of the specific binding of D2D3 to TROY and the ligand-receptor relationship

[0044] 1. Model building and preliminary screening:

[0045] Transgenic mice that specifically overexpress D2D3 in adipose tissue (hereinafter referred to as D2D3-Tg mice) were constructed. Figure 2A). Specifically, D2D3-Tg mice contain the coding sequence for D2D3 and, under the regulation of the AP2 promoter, overexpress the target protein D2D3 in mouse adipose tissue; the IgK signal peptide guides the release of D2D3 from adipocytes into the bloodstream.

[0046] like Figure 2 Immunofluorescence of B cells showed that D2D3 was present in the adipocytes of D2D3-Tg mice (molecular marker: Perilipin), but no D2D3 signal was found in the β cells of D2D3-Tg mice, indicating that D2D3 cannot enter β cells, but rather functions by binding to receptors on the surface of β cells.

[0047] To clarify the genetic changes encoding membrane proteins in D2D3-Tg mice, RNA-Seq analysis was performed on pancreatic islets from WT (wild-type) and D2D3-Tg mice. The results showed that the mRNA level of the membrane protein encoding gene TROY was significantly increased in D2D3-Tg mice. Figure 2 C).

[0048] 2. Verification of the interaction between D2D3 and TROY:

[0049] AlphaFold prediction and PyMOL analysis showed that there is a potential interaction interface between the TROY extracellular domain and D2D3. Figure 3 A, Figure 3 B).

[0050] Depend on Figure 3 As shown in Figure C, when HEK293T cells were transfected with expression plasmids of β1 integrin-Myc and Flag-TROY, as well as D2D3-Myc and Flag-TROY, co-immunoprecipitation (Co-IP) experiments using Myc antibody revealed that Flag-TROY was detected in the precipitation complex of D2D3-Myc and Flag-TROY. Similarly, when HEK293T cells were transfected with expression plasmids of Flag-K1 and D2D3-Myc, as well as Flag-TROY and D2D3-Myc, co-immunoprecipitation experiments using Flag antibody revealed that D2D3-Myc was detected in the precipitation complex of Flag-TROY and D2D3-Myc. This indicates that TROY and D2D3 bind intracellularly and interact with each other.

[0051] Subsequently, an in vitro pull-down experiment was performed using purified D2D3 and TROY proteins to further confirm that D2D3 and TROY directly interact. Figure 3 D).

[0052] 3. Combining kinetics and specificity :

[0053] Surface plasmon resonance (SPR) technology was used for analysis. D2D3 protein was immobilized on an SPR sensor chip CM5, and SPR experiments were performed using different concentrations of TROY protein as analytes. The equilibrium dissociation constants (K0) of the two proteins were measured. D The value is 48.44 nM. Figure 4 A, Figure 4 C Figure 4 D), indicating high affinity. On the other hand, after blocking TROY cells with an antibody that specifically recognizes the extracellular domain of TROY, the SPR experiment was performed again. At this point, the K values ​​of both were... D The value increased significantly to 1660 nM ( Figure 4 B. Figure 4 C Figure 4 D). This result indicates that D2D3 binds to the extracellular domain of TROY and the two interact directly, further establishing the ligand-receptor relationship between D2D3 and TROY.

[0054] Furthermore, Co-IP experiments with other members of the TNFRSF family (CD40, NGFR, RANK) showed that the binding of D2D3 to TROY is specific.

[0055] Example 2: Validation of TROY as a downstream molecule of D2D3 function

[0056] 1. Cell specificity:

[0057] Immunofluorescence staining of pancreatic tissue sections from WT and D2D3-Tg mice with insulin and TROY was performed. Figure 5 A). Furthermore, in pancreatic tissue sections from D2D3-Tg mice, immunofluorescence staining was performed using TROY antibody and insulin antibody (labeling β cells), glucagon antibody (labeling α cells), and somatostatin antibody (labeling δ cells), respectively. Figure 5 B), to determine the spatial location of TROY in different cell types of pancreatic islets.

[0058] Immunofluorescence results showed that the protein level of TROY was elevated in β cells of D2D3-Tg mice, and that TROY was mainly co-localized with insulin, while co-localizing with glucagon and somatostatin was less, confirming that TROY is specifically highly expressed in β cells of D2D3-Tg pancreatic islets.

[0059] 2. Functional rescue experiment:

[0060] Mice in which Troy was knocked out in WT mice were designated Troy KO mice. Figure 5C represents KO. Pancreatic islets were isolated from WT and TroyKO mice and treated with D2D3 protein. The effect of Troy deficiency on D2D3-mediated insulin secretion was determined using a glucose-stimulated insulin secretion assay (GSIS). Specifically, islets from WT and TroyKO mice were treated with vehicle (veh) and murine D2D3 protein (D2D3), respectively, and insulin secretion was measured at low glucose concentrations (2.8 mM) and high glucose concentrations (16.7 mM). Results are shown below. Figure 5 As shown in Figure C, it can be seen that the D2D3 protein significantly inhibited insulin secretion from WT islets, but had no significant effect on insulin secretion from Troy KO islets.

[0061] Furthermore, knocking out Troy in D2D3-Tg mice to create D2D3-Tg / Troy KO double mutant mice significantly improved fasting blood glucose and glucose tolerance compared to D2D3-Tg mice, restoring them to near WT levels. This result also demonstrates a functional relationship between D2D3 and Troy.

[0062] Example 3: Elucidating the mechanism by which the D2D3-TROY signaling axis impairs β-cell function

[0063] Pancreatic tissue sections were obtained from WT, D2D3-Tg, and D2D3-Tg / Troy KO mice. Immunofluorescence staining was performed using insulin antibody and Pdx-1 antibody, MafA antibody, and Nkx6.1 antibody, respectively, to determine the expression of each transcription factor. The results showed that, compared with WT mice, MafA expression in the pancreatic islets of D2D3-Tg mice was significantly reduced, while it was restored in D2D3-Tg / Troy KO mice. The regulation of MafA expression by D2D3-TROY was then validated in two ways:

[0064] 1. The direct regulatory pathway of D2D3-TROY on MafA:

[0065] MafA nucleoplasmic translocation analysis: The islets of pancreas of the WT, D2D3-Tg, and D2D3-Tg / Troy KO mice were treated with different concentrations (0, 10, 25 μM) of H2O2. Immunoblotting experiments were used to verify the translocation of MafA from the nucleus to the cytoplasm.

[0066] Localization of TROY and MafA in β cells: In MIN6 cells (mouse β cell line) treated with D2D3 protein, the localization of TROY and MafA in cells was determined by immunofluorescence assay. Considering that the intracellular domain of TROY may be cleaved, the applicant used an antibody that recognizes the intracellular domain of TROY for staining to clarify its nuclear translocation status;

[0067] Verification of the interaction between the TROY intracellular domain and MafA: A plasmid expressing the TROY intracellular domain and a plasmid expressing MafA were constructed and co-transfected into HEK293T cells. Immunoprecipitation experiments were performed to verify the interaction between the two.

[0068] Verification of D2D3-TROY Regulation of MafA Protein Stability: To verify the functional consequences of the above interaction, three groups were set up in MIN6 cells: Group 1 was treated with shRNA + PBS (control), Group 2 was treated with shRNA + D2D3 protein, and Group 3 was treated with shRNA-knockout Troy + D2D3 protein. After 24 hours of treatment, MIN6 cells in each group were treated with 10 μg / ml CHX for different time periods (0, 1, 2, 4 hours), and Western blotting was performed to verify the Troy knockout effect and to detect changes in MafA protein stability. The results showed that the stability of MafA protein in the shRNA + D2D3 protein treatment group was significantly lower than that in the control group, while the stability of MafA protein in the shRNA-knockout Troy + D2D3 protein treatment group was significantly improved compared to the shRNA + D2D3 protein treatment group, but slightly lower than that in the control group.

[0069] 2. The path by which D2D3-TROY indirectly regulates MafA through Kindlin-2:

[0070] Changes in the expression and localization of Kindlin-2 in disease models: Immunofluorescence staining and quantitative analysis of Insulin and Kindlin-2 were performed on pancreatic sections of mice of the above WT, D2D3-Tg and D2D3-Tg / Troy KO genotypes to clarify the changes in Kindlin-2 expression and nuclear translocation.

[0071] Validation of TROY Interference with the Kindlin-2-MafA Pathway: Kindlin-2 is known to bind to MafA and protect its stability. To verify whether TROY interferes with this protective mechanism, plasmids expressing both Kindlin-2 and MafA were transfected into HEK293T cells, with or without transfection of plasmids expressing the intracellular domain of TROY. The results showed that the presence of TROY weakened the interaction between Kindlin-2 and MafA, thereby disrupting the protection of MafA stability.

[0072] Example 3 demonstrates that the D2D3-TROY signaling axis promotes MafA protein degradation through direct interaction and interferes with the protective binding of Kindlin-2 to MafA via a dual molecular pathway, which synergistically leads to a decrease in MafA transcription factor levels and thereby impairs β-cell insulin secretion function.

[0073] Example 4: Elucidation of the mechanism by which the D2D3-TROY signaling axis inhibits β-cell proliferation

[0074] To elucidate how the D2D3-TROY signaling axis affects β-cell proliferation, its downstream pathways were analyzed by combining transcriptomics, protein level detection, and functional verification experiments.

[0075] 1. Transcriptomic analysis reveals suppression of proliferation-related signaling pathways:

[0076] RNA-Seq sequencing and bioinformatics analysis of the pancreatic islets of WT and D2D3-Tg mice revealed that the set of genes related to cell cycle progression in the pancreatic islets of D2D3-Tg mice was significantly enriched in downregulated pathways. Figure 6 A, Figure 6 B). Further analysis showed that the mRNA expression levels of several key genes in the PDGFRα-Ezh2 signaling pathway—a known key pathway regulating β-cell proliferation—including receptor Pdgfra, cyclins Ccnd1 and Ccnb2, and epigenetic regulator Ezh2, were significantly decreased in D2D3-Tg islets. Figure 6 C). These results suggest that the D2D3-TROY signaling axis may inhibit β-cell proliferation by suppressing the PDGFRα-Ezh2 pathway.

[0077] 2. Verify changes in proliferation markers at the protein level:

[0078] To confirm the above findings at the protein level, β-cell proliferation marker staining was performed on pancreatic islet tissues from WT, D2D3-Tg, and D2D3-Tg / Troy KO mice. Immunofluorescence staining and quantitative analysis showed that, compared with WT mice, the protein expression levels of nuclear proliferation antigen Ki67 and histone methyltransferase Ezh2 were significantly reduced in pancreatic islet β-cells of D2D3-Tg mice. Figure 6 D、 Figure 6 E). Importantly, in the islets of D2D3-Tg / Troy KO double mutant mice, the expression levels of both Ki67 and Ezh2 were significantly restored (E). Figure 6 D、 Figure 6 E). This result confirms that the inhibitory effect of D2D3 on β-cell proliferation is dependent on TROY, and that the D2D3-TROY signaling axis inhibits β-cell proliferation by regulating the PDGFRa-Ezh2 signaling pathway.

[0079] 3. Detection of downstream signaling pathway activation status:

[0080] To assess the activation status of the PDGFRα downstream signaling pathway, islets were isolated from the three mouse genotypes mentioned above and treated with their natural ligand PDGF-AA. The phosphorylation levels of Erk and Rb (pERK1 / 2 and pRB(Ser780)) were determined by Western blotting.

[0081] 4. Verification of β-cell proliferation function:

[0082] To definitively confirm the alterations in proliferative capacity at the functional level, islets of the three genotypes were treated with 50 ng / ml PDGF-AA for 48 hours, and simultaneously with 50 μM BrdU for 24 hours. Immunofluorescence assays were used to determine the uptake of BrdU by WT, D2D3-Tg, and D2D3-Tg / Troy KO islets. The results showed that BrdU uptake was significantly reduced in the D2D3-Tg group, while this reduction was mitigated in the D2D3-Tg / Troy KO group. These results demonstrate that the D2D3-TROY signaling axis indeed inhibits the proliferative response of β cells in response to growth factor stimulation, and that knocking out Troy effectively rescues this defect.

[0083] Example 5: Evaluation of the therapeutic effect of pentylene theophylline (PPF) targeting the D2D3-TROY axis

[0084] 1. PPF effectively inhibits D2D3-induced TROY protein expression:

[0085] To clarify the regulatory role of PPF on the target signaling axis, its effect on TROY protein levels was first examined in MIN6 cells (β cell line). MIN6 cells were stimulated with D2D3 protein to simulate a pathological state, followed by treatment with different concentrations (0, 5, 10, 20 μM) of PPF for 6 hours. Immunoblot analysis showed that PPF significantly reduced TROY protein levels in a concentration-dependent manner, with the inhibitory effects being particularly significant in the 10 μM and 20 μM PPF treatment groups. Figure 7 A). Therefore, 10 μM was selected as the optimal working concentration for subsequent experiments. Furthermore, time-course experiments showed that treatment of D2D3-stimulated MIN6 cells with 10 μM PPF resulted in a time-dependent decrease in TROY protein levels with increasing treatment time (0, 3, 6, 12, 24 hours). Figure 7 B). The above results demonstrate that PPF can effectively antagonize the upregulation of TROY by D2D3.

[0086] 2. PPF restores insulin secretion function suppressed by D2D3:

[0087] After clarifying the inhibitory effect of PPF on the target TROY, its functional effects were further evaluated. Under optimal treatment conditions (10 μM PPF, 24 hours), glucose-stimulated insulin secretion (GSIS) experiments were conducted in MIN6 cells, divided into a vector control group, a D2D3 treatment group, a D2D3+PPF co-treatment group, and a PPF treatment group. Results showed that in MIN6 cells, D2D3 treatment significantly reduced insulin secretion under high glucose stimulation. However, when co-treated with PPF, the inhibitory effect of D2D3 on insulin secretion was restored, showing a significant difference compared to D2D3 treatment alone. Figure 7 C). This result confirms that PPF can effectively salvage the insulin secretion function of β cells by intervening in the D2D3-TROY signaling axis.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various changes and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of TROY protein as a biomarker in the development of tools for diagnosing D2D3-related diabetes subtypes.

2. The use according to claim 1, characterized in that, The diagnosis includes comparing the expression level or activity level of TROY protein in the subject's sample with a reference value; an elevated level indicates that the subject has the diabetes.

3. The use according to claim 1 or 2, characterized in that, The D2D3-related diabetes subtype is characterized by the presence of specific binding between D2D3 and TROY in the patient's body, and / or increased TROY expression or activity mediated by D2D3.

4. Use of pentyl theophylline or its pharmaceutically acceptable salts, esters or solvates in the preparation of medicaments for the prevention and / or treatment of diabetes.

5. The use according to claim 4, characterized in that, The diabetes mellitus described is a D2D3-related diabetes mellitus subtype.

6. The use according to claim 5, characterized in that, The D2D3-related diabetes subtype is characterized by the presence of specific binding between D2D3 and TROY in the patient's body, and / or increased TROY expression or activity mediated by D2D3.

7. The use according to any one of claims 4 to 6, characterized in that, The theophylline exerts its therapeutic effect by downregulating TROY protein levels.

8. The use according to any one of claims 4 to 6, characterized in that, The theophylline is used to restore the insulin secretion function and / or proliferative capacity of pancreatic β cells.

9. The use according to claim 8, characterized in that, The restoration of insulin secretion function is related to the upregulation of MafA transcription factor expression or activity, and the restoration of proliferation capacity is related to the activation of the PDGFRα-Ezh2 signaling pathway.

10. A kit for diagnosing D2D3-related diabetes mellitus subtypes, the kit comprising reagents for detecting the expression and / or activity levels of TROY protein in a sample.

11. The reagent kit according to claim 10, characterized in that, The reagents include antibodies and / or antigen-binding fragments thereof capable of specifically binding to the TROY protein.

12. A reagent kit for precision medicine of diabetes, comprising: (a) a reagent for detecting the expression level of TROY protein in a sample; and (b) pentyl theophylline or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition for the prevention and / or treatment of diabetes, comprising: a therapeutically effective amount of pentylene or a pharmaceutically acceptable salt, ester or solvate thereof; and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 13, characterized in that, The diabetes mellitus described is a D2D3-related diabetes mellitus subtype.

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